Cellular localization of kinin B1 receptor in the spinal cord of streptozotocin-diabetic rats with a fluorescent [Nα-Bodipy]-des-Arg9-bradykinin

Abstract

Background

The kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines, bacterial endotoxins and hyperglycaemia-induced oxidative stress. In animal models of diabetes, it contributes to pain polyneuropathy. This study aims at defining the cellular localization of B1R in thoracic spinal cord of type 1 diabetic rats by confocal microscopy with the use of a fluorescent agonist, [Nα-Bodipy]-des-Arg9-BK (BdABK) and selective antibodies.

Methods

Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.). Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography. The B1R selectivity of BdABK was determined by assessing its ability to displace B1R [125I]-HPP-desArg10-Hoe140 and B2R [125I]-HPP-Hoe 140 radioligands. The in vivo activity of BdABK was also evaluated on thermal hyperalgesia.

Results

B1R was increased by 18-fold (mRNA) and 2.7-fold (binding sites) in the thoracic spinal cord of STZ-treated rats when compared to control. BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM). In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively. Intraperitoneal BdABK and des-Arg9-BK elicited dose-dependent thermal hyperalgesia in STZ-treated rats but not in control rats. The B1R fluorescent agonist was co-localized with immunomarkers of microglia, astrocytes and sensory C fibers in the spinal cord of STZ-treated rats.

Conclusion

The induction and up-regulation of B1R in glial and sensory cells of the spinal cord in STZ-diabetic rats reinforce the idea that kinin B1R is an important target for drug development in pain processes.

Background

Kinins are vasoactive peptides and central mediators acting through the activation of two G-protein-coupled receptors (R) denoted as B1 and B2 [1, 2]. The B2R is widely and constitutively expressed in central and peripheral tissues and is activated by its preferential agonists bradykinin (BK) and Lys-BK. The B1R is activated by the active metabolites des-Arg9-BK and Lys-des-Arg9-BK and has a low level of expression in healthy tissues. The latter receptor is upregulated after exposure to pro-inflammatory cytokines, bacterial endotoxins, and hyperglycaemia-induced oxidative stress [3–7].

An important role for kinin B1R has been postulated in nociception and pain [8–10]. B1R knock out mice are less sensitive to pro-inflammatory pain stimuli and to spinal sensitization [11–13]. B1R partakes to mechanical and/or thermal hyperalgesia induced by cytokines [14, 15] through peripheral protein kinase C activation [16] and in the formalin test [17, 18]. It also contributes to neuropathic pain after peripheral nerve injury [18–23] or after the induction of type 1 diabetes with streptozotocin (STZ) [24–27] and type 2 diabetes with high glucose feeding [7, 28, 29]. Thermal hyperalgesia was evoked by intraspinal stimulation of B1R in STZ-diabetic rats [9].

Basal expression of B1R was reported in the rat and human spinal cord dorsal horn as well as in rat dorsal root ganglion and small caliber primary sensory neurons [30–32]. Autoradiographic B1R binding sites are increased and distributed all over the grey matter of the spinal cord after peripheral nerve injury [22] and in models of diabetes [7, 29, 33]. This spatial distribution of B1R binding sites suggests that this receptor is not limited to primary sensory afferents but could also be present on spinal cord microglia and astrocytes.

To consolidate the role of B1R in pain polyneuropathy, its cellular distribution was investigated in the spinal cord of STZ-induced B1R with a newly developed fluorescent agonist named [Nα-Bodipy]-des-Arg9-BK (BdABK). The B1R selectivity of BdABK was determined by assessing its ability to displace B1R ([125I]-HPP-desArg10-Hoe 140) and B2R ([125I]-HPP-Hoe 140) radioligands by autoradiography. Moreover, the displacement of BdABK fluorescent labeling by B1R antagonists (R-715 and SSR240612) was assessed by confocal microscopy. We also investigated the in vivo activity of BdABK in comparison with its native agonist on thermal hyperalgesia in both STZ-treated and control rats. Appropriate selective antibodies were used in confocal microscopy to co-localize B1R on astrocytes, microglia and sensory C fibers in STZ-diabetic rats. The induction and overexpression of B1R in the spinal cord of STZ-diabetic rats was confirmed by qPCR and autoradiography. Experiments were achieved 4 days after STZ administration because previous studies showed that spinal cord B1R was maximally up-regulated and engaged in thermal hyperalgesia 2 days after STZ treatment [9, 33].

Methods

Animals and treatments

All research procedures and the care of the animals were in compliance with the guiding principles for animal experimentation as enunciated by the Canadian Council on Animal Care and were approved by the Animal Care Committee of our University. Male Sprague-Dawley rats (200–225 g, Charles River, St-Constant, Que., Canada) were housed two per cage, under controlled conditions of temperature (23°C) and humidity (50%), on a 12 h light-dark cycle and allowed free access to normal chow diet (Charles River Rodent) and tap water.

STZ treatment

Rats were used 5 days after their arrival and injected under low light with freshly prepared STZ (65 mg/kg; i.p.; Sigma-Aldrich, Oakville, ON, Canada). Age-matched controls were injected with vehicle (sterile saline 0.9%, pH. 7.0) [33]. Glucose concentrations were measured, with a commercial blood glucose-monitoring kit (Accusoft; Roche Diagnostics, Laval, Que., Canada), in blood samples obtained from the tail vein, in non-fasting animals, before STZ injection, and 4 days after treatment. Only STZ-treated rats whose blood glucose concentration was higher than 20 mM were considered as diabetic.

Synthesis of [Nα-Bodipy]-des-Arg9-BK

BdABK was synthesized using 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid succinimidyl ester (BODIPY® FL SE, Molecular Probes/Invitrogen Canada Inc, Burlington, ON; emission 510 nm) and des-Arg9-BK (Bachem Bioscience inc., King of Prussia, PA, USA). Des-Arg9-BK was solubilized in 100 mM NaHCO3 0.1 M (pH 8.4), at a concentration of 1 mg/ml and two equivalents of BODIPY® FL SE, solubilized in degassed dimethyl sulfoxide, at a concentration of 5 mg/ml was added. Completion of the reaction was achieved in 2 h, at ambient temperature, under continuous agitation. The fluorescent peptide was lyophilized and purified by C18 reverse-phase HPLC as previously described [34, 35]. The purity of the peptide was ≥ 98% as assessed by analytical HPLC (UV and fluorescence detection).

Tissue preparation for autoradiography and microscopy

Four days after injection of STZ, rats were anaesthetized with CO2 inhalation and then decapitated. Upper thoracic spinal cord (T3-T7) was removed and frozen in 2-methylbutane (cooled at -40°C following exposure to liquid nitrogen) and stored at -80°C. Few days later, spinal cords were mounted in a gelatin block and serially cut into 20-μm thick coronal sections with a cryostat. Thus the sections were thaw-mounted on 0.2% gelatin-0.033% chromium potassium sulfate-coated slides and kept at -80°C for 1 month to allow the adhesion of sections to the coverslip glasses.

Confocal microscopy

Slides preparation

On the day of experiments, sections were thawed at room temperature for 10 min to enhance sections adhesion. They were pre-incubated for 10 min in 25 mM PIPES-NH4OH buffer (pH 7.4) to allow degradation of endogenous kinins which could occupy receptors. Sections were exposed for 90 min to 50 μM BdABK. Thereafter, slides were washed twice (1 min) in PIPES and fixed with 4% para-formaldehyde [36]. Slides were washed three times (5 min) and then exposed to 1 M of glycine for 90 min to eliminate autofluorescence from aldehyde-fixed tissue. Tissues were permeabilized for 45 min with 0.1% Triton X-100.

Coverslip and microscopy

Slides were washed 3 times (5 min), mounted with coverslip, fixed with mowiol (12 h at room temperature) and stored at -4°C for 1 month or used in confocal microscopy.

SYBR green-based quantitative RT-PCR

Four days after injection of STZ, rats were anaesthetized with CO2 inhalation and then decapitated. The thoracic spinal cord (T1-T2) was isolated and approximately 10 mg of tissue were put in RNA later stabilization reagent (QIAGEN, Valencia, CA, USA). Total RNA was extracted from tissue according to the manufacturer's instructions. First-strand cDNA synthesized from 400 ng total RNA with random hexamer primers was used as template for each reaction with the QuantiTect Rev Transcription Kit (QIAGEN). SYBR Green-based real-time quantitative PCR using Mx3000p device for signal detection (Stratagene, La Jolla, CA, USA) was performed as described [43]. PCR was performed in SYBR Green Master mix (QIAGEN) with 300 nM of each primer. For standardization and quantification, rat 18S was amplified simultaneously. The primer pairs were designed by Vector NTI software and used [6] (Table 1).

PCR conditions were as follows: 95°C for 15 min, followed by 46 cycles at 94°C for 15 s, 60°C for 30 s and 72°C for 30 s. The cycle threshold (Ct) value represents the cycle number at which a fluorescent signal rises statistically above background [44]. The relative quantification of gene expression was analyzed by the 2-ΔΔCt method [45].

Quantitative autoradiography

The radioligands for kinin B1R, HPP-desArg10-Hoe140 (3-(4 hydroxyphenyl) propionyl-desArg9-D-Arg0 [Hyp3, Thi5, D-Tic7, Oic8]Bradykinin) and kinin B2R, HPP-Hoe140 (3-(4 hydroxyphenyl) propionyl-D-Arg0 [Hyp3, Thi5, D-Tic7, Oic8]Bradykinin) were synthesized and kindly provided by Dr Witold Neugebauer (Dept Pharmacology, University of Sherbrooke, Sherbrooke, Que., Canada). They were iodinated by the chloramine T method [46]. On the day of experiments, sections were incubated at room temperature for 90 min in 25 mM PIPES-NH4OH buffer (pH 7.4) containing: 1 mM 1,10-phenanthroline, 1 mM dithiothreitol, 0.014% bacitracin, 0.1 mM captopril, 0.2% bovine serum albumin (protease free) and 7.5 mM magnesium chloride in the presence of 200 pM of [125I]-HPP-desArg10-Hoe 140 or [125I]-HPP-Hoe 140 (specific activity: 2000 cpm/fmol or 1212 Ci/mmol) [29, 33]. Non-specific binding was determined in the presence of 1 μM of unlabeled B1R antagonist: R-715 (AcLys [D-βNal7, Ile8]des-Arg9-BK) [1] or of 1 μM of unlabeled B2R antagonist: Hoe 140 (Icatibant or JE 049, Jerini AG, Berlin, Germany) [47]. At the end of the incubation period, slides were transferred sequentially through four rinses of 4 min each in 25 mM PIPES (pH 7.4; 4°C) dipped for 15s in distilled water (4°C) to remove the excess of salts, and then air-dried. Kodak Scientific Imaging Films BIOMAX™ MR® (Amersham Pharmacia Biotech Canada) were juxtaposed onto the slides in the presence of [125I]-microscales and exposed at room temperature for 7 days. The films were developed (GBX developer) and fixed (GBX fixer). Autoradiograms were quantified by densitometry using an MCID™ image analysis system (Imaging Research, St. Catharines, ON, Canada). A standard curve from [125I]-microscales was used to convert density levels into fentomoles per milligram of protein [48]. Specific binding was determined by subtracting values of nonspecific binding from that of total binding.

Specificity of BdABK

To assess the specificity of BdABK for B1R, competition curves were performed in autoradiography by incubating 200 pM of [125I]-HPP-desArg10-Hoe 140 with increasing concentrations (10-10 to 10-6 M) of R-715 (selective B1R antagonist, kindly provided by Dr Domenico Regoli, Pharmacology, University of Ferrara, Italy), des-Arg9-BK (dABK, selective B1R agonist, Bachem Bioscience inc., King of Prussia, PA, USA) and BdABK. Moreover, competition curves were performed by incubating 200 pM of [125I]-HPP-Hoe 140 with increasing concentrations (10-10 to 10-6 M) of Hoe 140 (selective B2R antagonist) and BdABK. Each concentration of each competitor was tested on 4 sections per rat from 7 different rats. Those sections were exposed to the film, and total binding was calculated as described above. Moreover, the specificity of BdABK was determined in confocal microscopy by the displacement of fluorescent labeling with the addition of 10-5 M R-715 or SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4- [2R, 6S)-2,6 dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-ethylpropanamide hydrochloride] (kindly provided by Dr Pierre Carayon, Sanofi-Aventis, Montpellier, France) [18] to the incubation medium.

Microglial cell culture

Primary cell culture method

Mixed glial cultures were prepared following the protocol of McCarthy and de Vellis [49] with some modifications. Briefly, forebrains were dissected out from one litter of 2-day-old Sprague-Dawley rat pups and the meninges were stripped off before enzymatic and mechanical dissociation. For enzymatic dissociation, HBSS containing 0.25% trypsin (Gibco 15090-046) was used. The tissue-trypsin suspension was incubated for 20 min at 37°C in a water bath with intermittent shaking. After the waiting time for the trypsin digestion is over we added to the tissue-trypsin suspension a mixture of prewarmed DMEM/Dnase I (Sigma DN-25, Dnase I final concentration 0. 25 mg/ml) followed by an incubation for 4 min at 37°C. The resulting suspension was dispersed by a mild mechanical trituration which consisted in the passage through 18-, 22- and 25- gauge needles. This cell suspension was then filtred through 70 μm strainer (BD Falcon 352350). After extensive washs in prewarmed HBSS, these dissociated cells were resuspended and plated in 75-cm2 Falcon tissue-culture flasks (BD Biosciences) previously coated with 10 μg/ml poly-D-lysine (PDL). These mixed cells were growing at 37°C and 5% CO2 in DMEM (Gibco) supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 mg/ml). The media was changed every 2 or 3 days thereafter.

At 10 days-in-vitro, a confluent monolayer of astrocytes was apparent, on top of which oligodendrocyte precursor cells and a loosely attached layer of phase-bright microglia was obtained. Microglias were collected by shaking the flasks for 1 h at 200 rpm at 37°C and 5% CO2. Dislodged cells were resuspended and grown in culture medium for microglia [RPMI medium 1640 (Gibco) supplemented with 10% FBS, L-glutamine (1 mM), sodium pyruvate (1 mM), penicillin (100 units/ml), and streptomycin (100 mg/ml)]. The cells were allowed to adhere to the surface of PDL-coated coverslips (30 min at 37°C and 5% CO2), and nonadherent cells were rinsed off.

Microglia cells preparation for confocal microscopy

Briefly, confluent cells were exposed to 300 nM of BK for 24 h to induce B1R [50, 51]. Control cells were exposed to vehicle. After incubation with BdABK, cell were washed, then fixed and permeabilized with 100% methanol previously stored at -20°C. The fixed cells were then processed as described for immunostaining.

Thermal hyperalgesia

Thermal hyperalgesia was assessed according to the method described by Hargreaves et al., 1988 [52] with minor modifications. Briefly, rats were placed (unrestrained) within a Plexiglass enclosure on a transparent glass floor and allowed to acclimatize for 20–30 min. An infrared beam that constitutes the noxious heat stimulus (Plantar test, Ugo Basile, Italy) was moved beneath the plantar surface of the hind paw. Thermal nociceptive threshold was defined as the latency (seconds) between the heat stimulus (46°C) onset and the paw withdrawal using a feedback-controlled shut-down unit. A cut-off time of 33 s was used to avoid tissue damage. Each paw was tested three times alternatively at minimum intervals of 3 min between stimulation to avoid sensitization of the hind paw. The rats were trained on several days prior to testing B1R agonists. Thereafter, the thermal nociceptive threshold was assessed on 3 consecutive days as follows: day 1: baseline, saline and the first dose of des-Arg9-BK and BdABK (22.5 μg/kg); day 2: des-Arg9-BK and BdABK (225 μg/kg); day 3: des-Arg9-BK and BdABK (2250 μg/kg). Agonists were injected intraperitoneally at 1 h apart. This series of experiments was conducted in 3 control and 3 STZ-diabetic rats because the quantity of BdABK available for in vivo study was restricted. Thermal hyperalgesia was calculated as a percentage of the maximum possible effect (% MPE) according to the following formula: % MPE = (100 × (drug latency minus baseline latency)/(cut-off time minus baseline latency)) [9]. The baseline latency corresponds to the average of the first three measurements.

Statistical analyses

All data were expressed as means ± S.E.M. obtained from n rats. Statistical significance was determined with Student's t-test for unpaired samples or a one-way analysis of variance (ANOVA) followed by post-hoc Dunnett test for multiple comparisons. IC50 values were calculated by Graph Pad Prism 4.0 (GraphPad software, USA). Only probability (P) values less than 0.05 were considered to be statistically significant.

Results

B1R fluorescent labeling and selectivity of BdABK

Figure 1 illustrates B1R labeling with BdABK from low (i) to high (V) magnification (green dots) in dorsal horn of thoracic spinal cord of STZ-treated rats. As depicted in Figure 2, BdABK showed no labeling in control thoracic spinal cord (A), while the labeling of B1R was apparent in thoracic spinal cord of STZ-treated rats as revealed by green dots (B). Selectivity and specificity of the labeling were demonstrated by the absence of BdABK labeling in STZ-spinal cord sections when the B1R antagonists SSR240612 (D) and R-715 (E) were added at 10-5M.

Figure 1

B1R distribution in thoracic spinal cord of STZ-treated rats was shown by confocal microscopy with [Nα-Bodipy]-des-Arg9-BK. Shown are pictures from low (i) to high magnification (V) of the dorsal horn. Scale bars = 200, 50, 20, 5 and 2.5 μm, respectively from (i) to (V). Pictures are representative of a minimum of 4 sections per rat from 4 different STZ-diabetic rats.

[Nα-Bodipy]-des-Arg9-BK selectivity for B1R was evaluated by quantitative autoradiography. R-715 (selective B1R antagonist), des-Arg9-BK (selective B1R agonist) and [Nα-Bodipy]-des-Arg9-BK (fluorescent agonist of B1R) displaced in a concentration-dependent manner, from 10-10 to 10-6 M, the total binding of 200 pM [125I]-HPP-desArg10-Hoe 140 to B1R. Data are means ± SEM of 4 sections per rat from 7 different rats for each compound.

[Nα-Bodipy]-des-Arg9-BK affinity for B2R was evaluated by quantitative autoradiography. Increasing concentration (10-10 to 10-6 M) of Hoe 140 (selective B2R antagonist) displaced total binding of 200 pM [125I]-HPP-HOE-140 to B2R. In contrast, same concentrations of [Nα-Bodipy]-des-Arg9-BK (fluorescent B1R agonist) did not displace the B2R radioligand. Data are means ± SEM of 4 sections per rat from 7 different rats for each compound.

BdABK mediated in vivothermal hyperalgesia

The in vivo effect of BdABK on pain behavior was assessed by determining its ability to induce thermal hyperalgesia upon intraperitoneal injection in STZ-treated rats. As expected, BdABK and des-Arg9-BK had no significant effect on the nociceptive threshold in control rats, yet both agonists caused thermal hyperalgesia in STZ-diabetic rats at 0.225 and 2.25 mg/kg. These effects were dose-dependent and significant when compared to saline or control (Fig. 5). BdABK was however slightly but significantly less potent than des-Arg9-BK to induce hyperalgesia at the highest dose. As exemplified by des-Arg9-BK, this response peaked at 15 min post-injection and was reversible after 30 min (Fig. 6).

Figure 5

The ability of intraperitoneally injected [Nα-Bodipy]-des-Arg9-BK and its native agonist, des-Arg9-BK, to alter the paw withdrawal threshold in STZ-treated and control rats. Data represent maximal effects and are the average of 3 readings taken at 9, 12 and 15 min post-injection in 3 rats per group. Statistical comparison to control (*) and 2250 μg/kg des-Arg9-BK (+) are indicated by * + P < 0.05; ** P < 0.01; *** P < 0.001.

B1R mRNA expression assessed by qPCR

A low basal expression of kinin B1R mRNA was detected in the spinal cord of control rats (Fig. 7). This expression was significantly increased (18-fold) in the spinal cord of STZ-diabetic rats.

Figure 7

B1R mRNA expression in STZ-treated and control thoracic spinal cords was measured by quantitative real-time PCR. Data are means ± SEM of (3 to 6) rats. Statistical comparison with control is indicated by * P < 0.05.

Density of B1R binding sites assessed by quantitative autoradiography

As presented in Figure 8, quantitative in vitro autoradiography showed an increase density of specific B1R binding sites throughout the grey matter of the thoracic spinal cord in STZ-treated rats when compared to age-matched control spinal cord. B1R binding sites (2.4 fmol/mg protein) in spinal cord of STZ-treated rats were 2.7-fold greater than those measured in control rats (0.9 fmol/mg protein).

Figure 8

B1R binding sites in STZ-treated and control thoracic spinal cords were measured by quantitative autoradiography. Specific density of B1R binding sites are means ± SEM of (7 to 8) rats. Statistical comparison with control is indicated by *** P < 0.001.

B1R colocalized in primary cultured microglial cells

Figure 10 shows the colocalization of BdABK, TOPRO-3 and anti-IBA-1 in primary microglial cell culture. B1R was induced by a pre-treatment with 300 nM BK. About 95 ± 2% of the primary cell culture showed a positive labeling with anti-IBA-1 confirming cell purity. Data suggest that B1R can be induced in vitro on microglial cells.

B1R colocalized on sensory C fibers in thoracic spinal cord

Figure 11 shows the colocalization of BdABK, anti-TRPV1 and anti-CGRP in the thoracic spinal cord of STZ-treated rats. Data suggest that B1R and TRPV1 are co- localized on sensory C fibers in STZ-diabetic rats.

Discussion

This study is using a newly developed selective and high affinity fluorescent ligand enabling the cellular localization of B1R on unfixed tissue. It provides the first evidence that B1R is localized on microglial cells, astrocytes and sensory C fibers in the thoracic spinal cord of STZ-diabetic rats. This study also highlights the early upregulation of B1R (mRNA and binding sites) in the thoracic spinal cord of hyperglycaemic STZ-treated rats.

Diabetes induces B1R expression

STZ-diabetic rats provide an accessible model for studying the expression, the pharmacology and physiopathology of the B1R in the central nervous system. Pharmacological data showed that functional B1R was expressed in spinal cord of STZ-treated rats; its spinal activation led to sympathetically mediated increases of blood pressure and heart rate [53] and to thermal hyperalgesia [9]. Further autoradiographic and functional evidence for B1R induction was demonstrated in the lung [54], spinal cord [33], retina [6, 55] and brain [56] of STZ-diabetic rats. However, this is the first report on mRNA expression in thoracic spinal cord of STZ-diabetic rats by qPCR. Hyperglycaemia associated with type 1 diabetes can activate NF-κB [57] which is known to induce B1R [2, 3, 58]. Moreover, oxidative stress associated with diabetes was reported to be involved in the induction of B1R [6, 7, 29, 59].

[Nα-Bodipy]-des-Arg9-BK selectivity for B1R

Experiments by autoradiography confirm that BdABK is highly selective for B1R and does not bind to B2R. Indeed, BdABK failed to displace the B2R radioligand [125I]-HPP-Hoe-140 while it displaced the B1R radioligand, [125I]-HPP-desArg10-Hoe 140, with an IC50 of 5.3 ± 0.1 nM in thoracic spinal cord of STZ-treated rats. Results also evidenced that B2R binding sites were displaced by the selective antagonist, Hoe 140, with an IC50 value of 1.3 ± 0.1 nM while B1R binding sites were displaced by the natural B1R agonist, des-Arg9-BK (IC50 = 19 ± 0.2 nM) and by R-715, a selective B1R peptide antagonist (IC50 = 4.3 ± 0.2 nM). Comparison of IC50 values suggests that the affinity of the B1R agonist is increased by the addition of the Bodipy molecule. The stabilization of the N-terminus part of the peptide may contribute to prevent its degradation.

The reason for using 50 μM BdABK was based on preliminary study. The concentration of fluorescent probe needed to get a consistent labeling was higher than the IC50 value most likely because BdABK binds to B1R non-covalently and can be eliminated during the washout period of tissue sections. Signal amplification with radioactivity is also expected to be greater than that achieved with a fluorescent probe. BdABK showed no labeling in thoracic spinal cord of control rats which is in accordance with the inducible character of the B1R and its virtual absence in healthy tissues. The elimination of B1R labeling with BdABK after co-incubation with R-715 or SSR240612 confirms the specificity of the B1R fluorescent ligand.

Interestingly, BdABK maintained its biological activity as B1R agonist in vivo. Data obtained on the Hargreaves test revealed that BdABK was only slightly less potent than des-Arg9-BK to cause thermal hyperalgesia upon peripheral administration. This is consistent with the transient thermal hyperalgesia previously reported in the tail-flick test after intrathecal injection of des-Arg9-BK in rats made diabetics with STZ 24 h earlier [9]. Likewise, Gabra and Sirois [24] showed that intraperitoneal administration of des-Arg9-BK (400 μg/kg) in STZ-treated rats significantly reduced the paw withdraw threshold in the hot plate and tail-flick test.

Localization of B1R

B1R on microglial cells

Previous work by Noda and coworkers [50, 51] showed that B1R can be expressed in cultured rat microglia exposed to BK. We confirmed this result by using our fluorescent ligand in the same condition, thus providing additional evidence of its ability to bind B1R in a pure rat microglia model. BK acting via B2 receptors induces elevation of intracellular calcium leading to the phosphorylation and activation of NF-κB by protein kinase C [60]. NF-κB upregulates B1R upon binding to its nuclear promoter [2].

A recent study has demonstrated that B1R is involved in microglial migration toward rat brain lesion sites [61]. The presence of B1R on spinal microglial cells is in keeping with a recent study suggesting that activated dorsal horn microglia is a crucial component of STZ-induced tactile allodynia, mediated in part, by extracellular signal-regulated protein kinase signaling [62]. Importantly, the development of tactile and cold allodynia in a rat model of insulin-resistance was blocked by the B1R antagonist SSR240612 [28] and by two antioxidants (N-acetyl-L-cysteine and alpha-lipoic acid) known to prevent the induction of B1R [7, 29]. Taken together, these results suggest a critical role for microglial B1R in generation of tactile allodynia, a manifestation of pain polyneuropathy. It is possible that microglial B1R is also involved in STZ-induced thermal hyperalgesia as this response was abolished by B1R antagonists [5, 24, 27] and was absent in B1R knockout mice treated with STZ [26].

B1R on astrocytes

In addition, the present study provides the first evidence that thoracic spinal cord astrocytes bear the B1R in STZ-diabetic rats. Astrocyte B1R may represent another target for neuropathic or chronic pain. Emerging evidence suggests a critical role for astrocytes in the passage from acute to chronic and neuropathic pain. It seems that intracellular calcium level oscillation in astrocytes could spread through astrocytal network and thereby facilitate the formation of new synapses. These new synapses could establish neuronal contacts for maintaining and spreading pain sensation [63]. Moreover, astrocytes are known to release various inflammatory mediators that promote neuroimmune activation and can sensitize primary afferent sensory neurons contributing to development of neuropathic pain [64].

B1R on sensory C fibers

Immunohistochemical data showed the presence of B1R in DRG and superficial laminae of spinal cord dorsal horn [30–32]. Those studies suggested a basal expression of B1R in primary sensory C fibers of normal rat. This is consistent with the expression of B1R in sensory C fibers of STZ-treated rats as revealed by the co-localization of B1R, CGRP and TRPV1. Horowitz [65] described the crucial role of small A-delta and C fibers in generation of diabetic polyneuropathy and their sensitivity to hyperglycaemia. Ueda's studies [66, 67] support the hypothesis that the generation of neuropathic pain is related to alterations in gene and protein expression in primary sensory neurons which could contribute to demyelination of A-delta fibers through the down-regulation of myelin protein such as MBP, MPZ and PMP22. Demyelinated A-delta fibers sprout and synapse with A-beta fibers resulting in the enhancement of pro-nociceptive neurotransmitter release which generated allodynia. The presence of B1R on sensory C fibers is in agreement with an earlier pharmacological study that showed that the stimulation of B1R with an agonist in the spinal cord of STZ-diabetic rats provokes thermal hyperalgesia via the release of substance P [9].

Basal B1R expression in control rats

Authors failed to observe specific fluorescent labelling for B1R in normal rats which is rather consistent with the negligible level of B1R mRNA and binding sites. Moreover, intrathecal injection of B1R agonists or antagonists failed to cause behavioural, cardiovascular or nociceptive responses in control rats, suggesting that the basal expression of B1R is not functional in naïve rats [9, 53]. Thus the function of the B1R detected by immunohistochemistry in the spinal cord of rodents and human remains elusive. It is feasible that B1R in control animals is uncoupled to G protein as demonstrated for other G-protein-coupled receptors [68, 69]. Although it is possible that the immunological approach is more sensitive, we have evidence (unpublished data) showing that the commercially available B1R antibodies (M-19) from SantaCruz Biotechnologies (Santa Cruz, CA, USA) are not specific for immunohistochemical detection since B1R labeling persists in spinal cord isolated from B1R knockout mice. The latter B1R antibodies remain however suitable for Western blot analysis, suggesting that immunohistochemical studies reported with B1R antibodies remain to be validated with the appropriate controls in mutant mice.

Conclusion

[Nα-Bodipy]-des-Arg9-BK was found selective for B1R with an IC50 value of 5.3 ± 0.1 nM in the rat spinal cord. Furthermore, BdABK maintains its biological activity as agonist as evidenced by its ability to induce thermal hyperalgesia in STZ-treated rats. This new fluorescent ligand enabled the detection of B1R in primary microglial cell culture and on microglial cells, astrocytes and sensory C fibers in the thoracic spinal cord of STZ-diabetic rats. Because all these cells have been implicated in neuropathic pain, the induction and up-regulation of the B1R on these elements consolidate the idea that kinin B1R is an important target for drug development in pain processes.

Acknowledgements

This work was supported by Grant-in-aids from the Canadian Diabetes Association (OG-3-07-2428-RC) and Canadian Institutes of Health Research (MOP-79471). S.T. holds a Studentship from the FRSQ (Fonds de la Recherche en Santé du Québec). Authors acknowledge Ms Julie Desroches and Dr Pierre Beaulieu for giving us access to the Hargreaves Apparatus and to Mr Michel Lauzon for his expert assistance in confocal microscopy.